The detection of a wide variety of microorganisms in biological samples, food samples, potable water samples and pharmaceutical products is required for the effective treatment of disease and the maintenance of public health and safety. For example, platelet transfusions are often required for patients at risk from severe bleeding following myelosuppressive therapies, such as chemotherapy, or for patients with severe thrombocytopenia who undergo surgery. Platelet products are generally stored at room temperature (up to 5 days) and are thus susceptible to bacterial contamination and unrestricted bacterial growth. This is particularly relevant to platelet products that are produced by pooling of random donations. Patients that receive bacterially contaminated platelets can have severe reactions to the transfusion, especially when the patients are immunocompromised. It is estimated that 1 in 2,000 platelet bags are bacterially contaminated.
Traditional methods for evaluating bacterially contaminated platelets are based on culturing an aliquot of the platelet product for several days. Because of the time required for the culture, this procedure is often impractical for assessing products prior to transfusion given that during the several day test period any microbial colony forming units (CFU) would rapidly multiply. Only those patients appearing to have an adverse reaction to the transfusion will have their transfused product analyzed for contamination. Since this analysis occurs after the transfusion, the patients are at some medical risk. A rapid screening method for microbial contaminants would allow for screening of platelets for microbial contaminants prior to transfusion, minimizing the risks of patient infection. Despite this need, no such rapid screening method is currently commercially available.
Conventional techniques for the detection of microorganisms in suspect samples often require the growth of the microorganism in order to increase the cell numbers. Following a one to five day growth period in nutrient media, the microorganisms are detected using a wide variety of detection methods. Fluorescence labeled detection markers have increased sensitivity over conventional calorimetric marks and offer the possibility of reduced analysis times because they require less cell numbers to register a positive result. For example, a fluorescent derivative of polymyxin has been used to show binding to particular species of microorganisms. (Newton, B. A. J. gen. Microbiol. 1955) This compound was used to localize polymyxin binding in fractionated cellular components. Pure cultures of the target organisms were grown in enriched media to ensure high cell counts of target cells. Viewing the fluorophore required washing and isolation of the target cells to separate the cells from the background fluorescence and to eliminate from the target cells biological and other compounds that would interfere with the absorption or emission wavelengths of the fluorescent derivative.
More recently, fluorometric methods have been developed using a wide variety of combinations of binding agents and detectable labels. In these assays the binding agent is labeled with a fluorescent marker. The binding agent binds to the microorganism and the presence or absence of the microorganism can be determined using various fluorometric measuring devices, for example fluorescence microscopy or flow cytometry. Examples of such binding agents include antibodies, DNA/RNA probes, phage and other agents that bind to some target cell component.
Despite these improvements in technique, rapid fluorometric techniques still suffer from numerous problems. An initial problem is inadequate sensitivity and/or specificity. Heretofore used fluorescent detection methods for microorganisms which are species and sometimes genus specific can not screen for a wide variety of microorganism species in a suspect sample. For example, antibodies and nucleic acid probes with a fluorescent label are widely used, but only to screen for specific pathogens. Typical examples of such tests include specific fluorometric assays for Salmonella using antibodies and assays employing nucleic acid probes for E. coli 0157. To screen for all microbial contaminants in a given biological sample would require numerous assays, one for each microorganism of interest. These assays also lack adequate sensitivity. Their reported detection limits are often at 10,000 CFU/mL or more. The introduction of the polymerase chain reaction (PCR) and similar amplification techniques have greatly improved these detection limits; however they can no longer be regarded as rapid methods as they often take up to one day to complete.
Rapid fluorometric screening methods which employ a direct stain have been developed and are widely used. They too have several problems. First, direct staining often lacks sensitivity. In addition these assays can also lack specificity. Direct methods employ a stain which binds to some component or cell structure of the microorganism which causes it to take on a fluorescent signal. Wheat germ agglutinin, for example, binds to a wide variety of gram positive cell walls including red blood cells. When these compounds are conjugated with a fluorescent label they may be used for rapid detection of gram positive organisms (U.S. Pat. No. 5,137,810). Like many similar tests sensitivity is increased by removing cells from their native environment, washing the cells, and detecting the gram positive organisms on a solid support. Direct fluorometric DNA staining reagents have also been developed and are widely used. Typical of these is acridine orange. This compound is taken up into the cell where it binds specifically with nucleic acid (DNA and RNA) resulting in a characteristic fluorescent emission. These stains will stain all cells that contain DNA and extensive pre-treatment and filtration techniques have been developed to improve the specificity of these assays. More information could be gathered more rapidly with an assay capable of directly targeting within a biological sample a wide variety of microorganisms, with the ability of the assay to specifically target either specific species or broader classes of microorganisms.
It is the object of the invention to provide a method to screen biological samples and rapidly detect low levels of microorganisms. It is another object to provide a method to detect microorganisms that does not require culturing steps and minimizes the purification steps required by this method. It is a further object to provide an assay for microorganisms that can alternative- ly be either specific to a given species or genus of microbe or broad spectrum, capable of detecting an entire class of microorganisms.
Another object of the invention is to describe a novel antibiotic/fluorescent dye conjugate. The fluorescent marker used in this compound should be detectable when present in a biological sample.